Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease of the connective tissues, for which there are no satisfactory treatment options. It is a multisystem disorder that can cause fibrosis of the skin and internal organs, including lungs, heart, kidneys and the gastrointestinal tract. SSc afflicts 100,000-150,000 people in the US alone, with approximately 1500 new cases diagnosed each year. Current management of SSc entails the use of immunosuppressives such as methotrexate and cyclophosphamide, which are cytotoxic and of limited efficacy. Although the pathology and progression of SSc have been well described, the etiology of the disease is still not clear. The hallmark of SSc and other fibroses is the transition of normal fibroblasts into myofibroblasts, which are characterized by expression of alpha- smooth muscle actin (-SMA) and production of collagen-rich extracellular matrix (ECM). This pro-fibrotic fibroblast-to-myofibroblast transitin can be induced by a number of extracellular stimuli, most of which appear to depend on the RhoA signaling pathway. This pathway leads to the release of G-actin-bound myocardin- related transcription factor (MRTF), subsequent accumulation of MRTF in the nucleus, binding of MRTF to serum response factor (SRF) on the serum response element (SRE) promoter, and consequent upregulation of expression of a number of pro-fibrotic genes, including ACTA2, the gene encoding for a-SMA. We have discovered and developed a novel class of small molecule inhibitors of the Rho/MRTF/SRF gene transcription pathway. A lead compound from this series (CCG-203971) inhibits expression of a-SMA in both TGF-b- stimulated normal dermal fibroblasts as well as skin myofibroblasts from SSc patients. Despite this promising in vitro activity, CCG-203971 has modest potency and poor pharmacokinetic (PK) properties that limit its use as a probe for studying the effects of inhibiting the Rho/MRTF/SRF pathway in in vivo models of fibrosis. The overarching hypothesis of this proposal is that pharmacological inhibition of Rho/MRTF/SRF-mediated gene transcription will be an effective new treatment strategy for SSc. Our approach will be to: 1) optimize the lead CCG-203971 for both potency and physicochemical properties predictive of good PK; 2) evaluate the best new analogs for in vivo PK; and 3) evaluate optimal probes for efficacy in two complementary mouse models of fibrosis. This proposal is innovative in that it will attempt to treat or prevent fibrosis through disruptionof the Rho/MRTF/SRF signaling pathway at a point that is downstream of and common to multiple extracellular pro-fibrotic stimuli. This work is significant in that it will attempt for the first ime to directly prevent the fundamental myofibrblast differentiation that underlies fibrosis. Finally, the ultimate impact of this work will be progress toward the development of an entirely new therapy for SSc and potentially many other fibrotic diseases.